FUS is an RNA‐binding protein (RBP) with a prion‐like domain (PrLD) that condenses into functional liquids, which can aberrantly phase transition into solid aggregates comprised of pathological fibrils connected to neurodegenerative disease. How cells prevent aberrant phase transitions of FUS and other disease‐linked RBPs with PrLDs is poorly understood. In this issue of The EMBO Journal, Monahan et al (2017) establish that phosphorylation of specific serine and threonine residues in the FUS PrLD inhibits aberrant phase separation and toxicity.

(A) Domain architecture of FUS. FUS harbors an N‐terminal PrLD (residues 1–239), a RNA‐recognition motif (RRM; residues 285–370), two RGG domains (residues 371–421 and 453–500) separated by a zinc‐finger domain (ZNF; residues 422–452), and a C‐terminal PY‐nuclear localization sequence (residues 501–526). (B) The uncharged, polar PrLD enables FUS to undergo liquid–liquid phase separation (LLPS). In these condensed liquid droplets, the FUS PrLD initially remains intrinsically disordered, but the high local concentration of PrLDs promotes their nucleation into cross‐β fibrils. As stable cross‐β fibrils begin to dominate the droplet there is a shift from the liquid state to a hydrogel state and ultimately to a solid aggregate comprised of pathological fibrils. DNA‐PK phosphorylates multiple sites in the FUS PrLD, which permits LLPS of full‐length FUS but prevents the primary nucleation of cross‐β fibrils in the liquid droplet and the aberrant phase transition to pathological fibrils.

FUS shuttles between the nucleus and cytoplasm, but is predominantly localized to the nucleus where it performs critical functions in transcription, pre‐mRNA splicing, RNA processing, and DNA repair (Harrison & Shorter, 2017). For many of these modalities, FUS likely functions in the concentrated and specialized microenvironment of a membraneless organelle or RNP granule (Patel et al, 2015; Harrison & Shorter, 2017). Importantly, the uncharged, polar PrLD enables FUS to undergo liquid–liquid phase separation (LLPS), which contributes to the biogenesis of these membraneless organelles (Fig 1B) (Burke et al, 2015; Murakami et al, 2015; Patel et al, 2015). In these condensed liquid states, the FUS PrLD adopts a molten, disordered conformation just as in the dispersed soluble state, but eventually the high local concentration of PrLDs promotes their nucleation into cross‐β fibrils (Fig 1B) (Burke et al, 2015; Patel et al, 2015; Kato & McKnight, 2017). This aberrant phase transition is accompanied by a shift in the material state of the droplet from liquid to a gel as cross‐β FUS fibrils begin to dominate the droplet (Fig 1B) (Patel et al, 2015; Kato & McKnight, 2017). Initially, short cross‐β FUS fibrils may be labile and reversible (Kato & McKnight, 2017), but ultimately the droplet converts to a deleterious solid state comprised of pathological fibrils (Murakami et al, 2015; Patel et al, 2015). Disease‐linked mutations in the FUS PrLD accelerate this aberrant phase transition from liquid drops to pathological fibrils (Murakami et al, 2015; Patel et al, 2015). In ALS and FTD, this process likely occurs in cytoplasmic stress granules, which may convert into pathological aggregates (March et al, 2016). How cells prevent aberrant phase transitions of FUS and other disease‐linked RBPs with PrLDs is poorly understood. Monahan et al (2017) now establish that phosphorylation of specific serines and threonines in the FUS PrLD inhibits formation of intra‐ and intermolecular contacts that drive aberrant phase separation and toxicity.

Collectively, these advances suggest that strategies to increase FUS PrLD phosphorylation could have therapeutic utility for ALS and FTD, which can now be assessed in more complex disease models including fly, mouse, and patient‐derived neurons. These exciting studies also inspire several questions. For example, the precise role of FUS phosphorylation in physiology and pathology remains unknown. Nevertheless, introduction of multiple negatively charged residues into PrLDs appears to be a powerful mechanism to alter the syntax of self‐assembly and promote liquid states over deleterious solid states (Monahan et al, 2017). However, the FTD‐linked FUS variant, G156E, exhibits accelerated aberrant phase separation (Patel et al, 2015), which raises the possibility that phosphorylation of a single serine or threonine in the FUS PrLD could promote aberrant phase separation. Thus, it is critical that the FUS PrLD gets phosphorylated at multiple sites to mitigate toxicity (Monahan et al, 2017). It also remains unclear whether phosphorylation of the FUS PrLD can drive the dissolution of preformed FUS fibrils and return the aberrant solid aggregates that accumulate in disease to a liquid droplet phase or to soluble monomeric species. It will be important to determine whether key serine and threonine residues that may be buried in the FUS fibril core are accessible to protein kinases. An interesting possibility is that protein kinases might work in conjunction with protein disaggregases to rapidly phosphorylate newly disaggregated FUS and thereby prevent reaggregation (Yasuda et al, 2017).

Another question relates to DNA‐PK, which is localized to the nucleus and likely prevents aberrant FUS phase transitions in this compartment. However, in ALS and FTD, FUS accumulates in the cytoplasm of degenerating neurons. Thus, a goal would be to increase FUS PrLD phosphorylation in the cytoplasm, which may require targeting another kinase or perhaps inhibiting a phosphatase. Precisely how increased FUS PrLD phosphorylation might be elicited pharmacologically is also unclear. Whether phosphorylation of the FUS PrLD negatively regulates FUS functions in transcription, pre‐mRNA splicing, and RNA processing will also be critical to determine as it could be disadvantageous to exacerbate FUS loss of function in disease. Nonetheless, phosphorylation of the FUS PrLD effectively counters toxicity due to cytoplasmic gain of toxic function (Monahan et al, 2017), which is emerging as an important mechanism underpinning neurodegeneration in mouse models (Sharma et al, 2016). Finally, serine, tyrosine, and threonine occur commonly in PrLDs (March et al, 2016). Thus, it will be enlightening to establish whether specific phosphorylation events in PrLDs of other disease‐linked RBPs inhibit aberrant phase separation, which might also be targeted therapeutically. Regardless, the groundbreaking discoveries made by Monahan et al (2017) establish phosphorylation of the FUS PrLD as an intriguing new therapeutic strategy for diverse FUS proteinopathies.